Water and electrolyte content and distribution in tissues of thermally-acclimated rainbow trout, Salmo gairdneri

The primary objective of this investigation was that of providing a comprehensive tissue-by-tissue assessment of water-electrolyte status in thermally-acclimated rainbow trout, Salmo gairdneri. To this end levels of water and the major ions, sodium, chloride and potassium were evaluated in the plasma, at three skeletal muscle sites, and in cardiac muscle, liver, spleen, gut and brain of animals acclimated to 2°, 10° and 18°C. The occurrence of possible seasonal variations in water-electrolyte balance was evaluated by sampling sununer and late fall-early winter populations of trout. On the basis of values for water and electrolyte content, estimates of extracellular and cellular phase volumes, cellular electrolyte concentrations and Nernst equilibrium potentials were made. Since accurate assessment of the extracellular phase volume is critical in the estimation of cellular electrolyte concentrations and parameters based on assumed cellular ion levels, [14 C]-polyethylene glycol-4000, which is assumed to be confined to the extracellular space, was employed to provide comparisons with various ion-defined spaces (H20~~s, H20~~/K and H20~~s). Subsequently, the ion-defined space yielding the most realistic estimate of extracellular phase volume for each tissue was used in cellular electrolyte calculations. Water and electrolyte content and distribution varied with temperature. Tissues, such as liver, spleen and brain appeared to be the most thermosensitive, whereas skeletal and cardiac muscle and gut tissue were less influenced. 'Summer' series trout appeared to be more capable of maintaining their water- electrolyte balance than the ~fall-winter' series animals. i The data are discussed in terms of their possible effect on maintenance of appropriate cellular metabolic and electrophysiological functions

Hsp27 and axonal growth in adult sensory neurons in vitro

BACKGROUND: Neurite growth can be elicited by growth factors and interactions with extracellular matrix molecules like laminin. Among the targets of the signalling pathways activated by these stimuli are cytoskeletal elements, such as actin, tubulin and neurofilaments. The cytoskeleton can also be modulated by other proteins, such as the small heat shock protein Hsp27. Hsp27 interacts with actin and tubulin in non-neuronal cells and while it has been suggested to play a role in the response of some neurons to injury, there have been no direct studies of its contribution to axonal regeneration. RESULTS: We have investigated neurite initiation and process extension using cultures of adult dorsal root ganglion (DRG) sensory neurons and a laminin stimulation paradigm. Employing confocal microscopy and biochemical analyses we have examined localization of Hsp27 at early and later stages of neurite growth. Our results show that Hsp27 is colocalized with actin and tubulin in lamellopodia, filopodia, focal contacts and mature neurites and growth cones. Disruption of the actin cytoskeleton with cytochalasin D results in aberrant neurite initiation and extension, effects which may be attributable to alterations in actin polymerization states. Inhibition of Hsp27 phosphorylation in our cultures results in an atypical growth pattern that may be attributable to an effect of pHsp27 on the stability of the actin cytoskeleton. CONCLUSION: We observed colocalization of the phosphorylated and non-phosphorylated forms of Hsp27 with actin and tubulin in both very early and later stages of neurite growth from cultured adult DRG neurons. The colocalization of Hsp27 and pHsp27 with actin in lamellopodia and focal contacts at early stages of neurite growth, and in processes, branch points and growth cones at later stages, suggests that Hsp27 may play a role in neuritogenesis and subsequent neurite extension, and potentially in the patterning of this growth. Hsp27 has been reported to play a key role in modulating actin cytoskeletal dynamics as an actin-capping protein in non-neuronal cells. Our results suggest that this may also be the case in neurons and support a role for Hsp27 in neurite outgrowth via its phosphorylation state-dependent interactions with actin

Data from: Extracellular matrix-associated gene expression in adult sensory neurons cultured on laminin substrates

Background: In our previous investigations of the role of the extracellular matrix (ECM) in promoting neurite growth we have observed that a permissive laminin (LN) substrate stimulates differential growth responses in subpopulations of mature dorsal root ganglion (DRG) neurons. DRG neurons expressing Trk and p75 receptors grow neurites on a LN substrate in the absence of neurotrophins, while isolectin B4-binding neurons (IB4+) do not display significant growth under the same conditions. We set out to determine whether there was an expression signature of the LN-induced neurite growth phenotype. Using a lectin binding protocol IB4+ neurons were isolated from dissociated DRG neurons, creating two groups - IB4+ and IB4-. A small-scale microarray approach was employed to screen the expression of a panel of ECM-associated genes following dissociation (t=0) and after 24 hr culture on LN (t=24LN). This was followed by qRT-PCR and immunocytochemistry of selected genes. Results: The microarray screen showed that 36 of the 144 genes on the arrays were consistently expressed by the neurons. The array analyses showed that six genes had lower expression in the IB4+ neurons compared to the IB4- cells at t=0 (CTSH, Icam1, Itgβ1, Lamb1, Plat, Spp1), and one gene was expressed at higher levels in the IB4+ cells (Plaur). qRT-PCR was carried out as an independent assessment of the array results. There were discrepancies between the two methods, with qRT-PCR confirming the differences in Lamb1, Plat and Plaur, and showing decreased expression of AdamTs1, FN, and Icam in the IB4+ cells at t=0. After 24 hr culture on LN, there were no significant differences detected by qRT-PCR between the IB4+ and IB4- cells. However, both groups showed upregulation of Itgβ1 and Plaur after 24 hr on LN, the IB4+ group also had increased Plat, and the IB4- cells showed decreased Lamb1, Icam1 and AdamTs1. Further, the array screen also detected a number of genes (not subjected to qRT-PCR) expressed similarly by both populations in relatively high levels but not detectably influenced by time in culture (Bsg, Cst3, Ctsb, Ctsd, Ctsl, Mmp14, Mmp19, Sparc. We carried out immunohistochemistry to confirm expression of proteins encoded by a number of these genes. Conclusions:Our results show that 1B4+ and IB4- neurons differ in the expression of several genes that are associated with responsiveness to the ECM prior to culturing (AdamTs1, FN, Icam1, Lamb1, Plat, Plaur). The data suggest that the genes expressed at higher levels in the IB4- neurons could contribute to the initial growth response of these cells in a permissive environment and could also represent a common injury response that subsequently promotes axon regeneration. The differential expression of several extracellular matrix molecules (FN, Lamb1, Icam) may suggest that the IB4- neurons are capable of maintaining /secreting their local extracellular environment which could aid in the regenerative process. Overall, these data provide new information on potential targets that could be manipulated to enhance axonal regeneration in the mature nervous system

ECM IB4+ t=24LN expressed genes reps1-6

Data showing genes detected on microarrays for IB4-expressing dorsal root ganglion neurons at t=24LN (sampled after 24 hr culture on laminin-coated substrate). See Read-me file for details. Only genes detected above background are listed; of 144 ECM genes on the arrays, only 36 genes were expressed by both neuron populations

ECM IB4+ t=0 expressed genes reps1-7

Data showing genes detected on microarrays for IB4-expressing dorsal root ganglion neurons at t=0 (smapled immediately after selection protocol). See Read-me file for details. Only genes detected above background are listed; of 144 ECM genes on the arrays, only 36 genes were expressed by both neuron populations

ECM IB4neg t=0 expressed genes reps1-7

Data for non-IB4 expressing DRG neurons at t=0; these were sampled immediately after the selection protocol that removes the IB4-expressing neurons, leaving the IB4-negative neurons behind. Only genes detected as being expressed are presented.Of 144 ECM genes on the arrays, 36 genes were expressed by both neuron populations

ECM IB4neg t=24LN expressed genes reps1-6

Data for non-IB4 expressing DRG neurons at t=24LN; these were sampled after 24 hr culture on laminin-coated substrate. Only genes detected as being expressed are presented.Of 144 ECM genes on the arrays, 36 genes were expressed by both neuron populations

List of genes on microarray ORN-013.2

Complete list of genes on GEArray ORN-013.

List of genes on microarray ORN-013

Complete list of genes on GEArray ORN-01

HspB1 is associated with F-actin.

<p>Representative blots showing precipitation of F-actin complexes (with biotinylated phalloidin, A–B) and IPs of HspB1 (C–D) from total cellular lysates (A, C) or the cytoskeletal pellet fraction (B, D). PC12 cells were treated as described in the Methods. Cell cultures were exposed to 10 µM SB203580 for 1 hr, after which cultures were either incubated for an additional 30 mins, or stressed with heat shock at 42°C for 30 mins; control cells were not treated. Immediately after treatments, cells were collected, lysed with actin stabilization buffer; samples were separated for analysis as total cell lysate or further fractionated into the TritonX-100 insoluble cytoskeletal pellet. Samples were incubated with biotinylated-phalloidin followed by precipitation of the captured complexes with streptavidin-linked magnetic beads. Precipitated fractions were then subjected to SDS-PAGE and sequentially immunoblotted to detect pHspB1, total HspB1 and actin. Pulldown of F-actin also captures HspB1 in both the cell lysates (A) and the cytoskeletal fraction (B); similarly the HspB1 IPs also bring down actin and this is enhanced in the cytoskeletal fraction.</p